Defining the regulators of stem cell fate is critical in deriving lineage-specified populations for tailored regenerative therapy. Beyond established genetic/epigenetic control of cell identity, emerging evidence indicates that the way stem cells use energy and metabolites determine their fate. To maintain tissue regenerative capacity, stem cells must transit between distinct states from active proliferation/self-renewal to differentiatio, each imposing unique bioenergetic demands. To enable transition between distinct states, metabolic plasticity prioritizes biochemical pathways to establish a balance between catabolism, the process of breaking down substrates to produce energy, and anabolism, the process of constructing macromolecules. The overall goal of this proposed research is to understand how plasticity in energy metabolism regulates cell fate decisions and how specific metabolic pathways support stage-specific stem cell function. The K99 phase of this award will focus on defining the specific mitochondrial dependent components and metabolic pathways essential for nuclear reprogramming of somatic cells back to the pluripotent state. This will be accomplished by comparing bona fide induced pluripotent stem cells versus their parental fibroblasts using fundamental mitochondrial biology techniques and high throughput metabolomics/metabolic flux analysis, and validating the impact of identified pathways on nuclear reprogramming using mitochondrial deficient cells and enzyme- specific inhibition. Having developed this expertise in mitochondrial biology and high throughput metabolomics and metabolic flux analysis, the R00 phase of the award will focus on applying these new technologies to a cardiac differentiation protocol that allows isolation of developmental stages during cardiogenesis in order to define the specific mitochondrial complexes and metabolic reprogramming that fuels lineage specification, using cardiac differentiation as a model system. Collectively the K99 and R00, by defining the distinct components of mitochondrial function necessary for initiation of metabolic reprogramming during induction of pluripotency and lineage specification, will fundamentally advance our current knowledge of stem cell metabolism and how it impacts cell fate. Application of this knowledge will enable development of metabolic strategies for generation of tissue-specific cell populations for regenerative therapy, with potential translation to identify new targets based upon energy metabolism to prevent degeneration or elicit rejuvenation in chronic disease. The synergy of the candidate's research experience and outstanding current environment has laid the foundation for the present application in the field of stem cell metabolism and cell fate decisions. His graduate work built upon initial undergraduate training in biochemistry that provided a comprehensive knowledge base in energy metabolism and cardiovascular pathophysiology, which was enhanced with training in stem cell biology and metabolomics profiling during the initial post-doctoral training. The K99 phase of this award will be based at Mayo Clinic, where the core mission focuses on research and education aimed to advance the science of medicine, improve disease prevention and treatment, and prepare next generations of health care professionals. The scientific proposal will benefit from Mayo Clinic's cutting edge technical platforms and core resources, while career development will be facilitated by tailored curriculum established by the mentor, leveraging the career development instruments of Mayo Clinic. The candidate's short-term goals include publication of current and proposed manuscripts on the mitochondrial/metabolic determinants of nuclear reprogramming (K99 phase), establishment of model systems required for the proposed projects and acquisition of comprehensive skill sets to dissect mitochondrial and metabolic function in the setting of decoding stem cell fate decisions. Completion of these goals will facilitate the candidate's long-term career goal of establishing an independent and extramurally funded research program to examine how plasticity in energy metabolism regulates cell fate decisions and how specific metabolic pathways support stage-specific stem cell function (R00 phase). Career development will be fostered through interaction with the mentor, Dr. A. Terzic (Director, Mayo Clinic Center for Regenerative Medicine) and the advisory committee composed of Drs. J. Burnett Jr. (Former Director, Mayo Clinic Heart and Lung Research Center), S. Nair (Director, Mayo Clinic Comprehensive Metabolomics Center), F. Prendergast (Guggenheim Professor of Biochemistry and Molecular Biology), and J. van Deursen (Chair, Department of Biochemistry and Molecular Biology). Emphasis will be placed on acquisition of new skills and methodology, specifically in the areas of mitochondrial biology, metabolomics, metabolic flux analysis and systems biology to complement the current expertise in stem cell biology and NMR based metabolomics. This technical proficiency paired with the conceptual innovation outlined in the scientific proposal provides a launchpad to embark on an independent research career in the emerging domain of stem cell metabolism.